Production of Secondary Metabolites in Plant Cell Cultures - American

26 Production of Secondary Metabolites in Plant Cell Cultures

Downloaded by UNIV OF ARIZONA on September 10, 2015 | http://pubs.acs.org Publication Date: August 25, 1986 | doi: 10.1021/bk-1986-0317.ch026

Robert J. Whitaker, George C. Hobbib, and Leslie A. Steward DNA Plant Technology Corporation, 2611 Branch Pike, Cinnaminson, NJ 08077 The initial enthusiasm for tapping the vast synthetic potential of cultured plant cells has largely given way to the realization that much needs to be learned about the biochemical and genetic regulation of plant secondary metabolism before cost-effective, industrial-scale production becomes feasible. However, the rapidly emerging technologies of plant tissue culture biotechnology are poised to have significant impact on advancing the commercialization of valuable plant secondary metabolites. A key to economically sound production is clearly the induction and selection of high-yielding cell cultures. Somaclonal variation has proven to be a powerful tool for uncovering useful genetic variation for the improvement of agriculturally important crop plants. In a similar fashion, somaclonal variation technology can be used to induce high-yielding cell cultures. Once producing variants have been selected, precise definition of growth and production media will further enhance production and maintain the genetic stability of producing cultures. Plants are a valuable source of a vast array of chemical compounds including flavors, fragrances, pigments, natural sweeteners, industrial feedstocks, antimicrobials, and pharmaceuticals. These compounds belong to a rather broad group of metabolites collectively referred to as secondary products. The precise physiological function of secondary products has been a topic of debate among researchers. However, it seems clear that secondary products have not evolved to perform vital physiological functions, in the same manner as primary compounds like amino acids or nucleic acids, but rather seem to serve as a chemical interface between the producing plants and their surrounding environment. For example, plants may produce secondary products to ward off potential predators, attract pollinators, or combat infectious diseases (1). A number of plant species commonly sought for their secondary products are native to very remote and sometimes politically unstable 0097-6156/ 86/ 0317-0347506.00/ 0 © 1986 American Chemical Society American Chemical Society Library 1155 18th St., N.W.

Washington, D £ 20036 In Biogeneration of Aromas; Parliment, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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geographic areas of the world. A d d i t i o n a l l y as with any plant grown in the environment, the plants that produce valuable secondary products are subjected to a variety of c l i m a t i c stresses that can ultimately determine the level and the quality of production. These factors greatly impinge upon the reliability of the particular product which, of course, is a major concern for industrial processors. Indeed, wild fluctuations in a v a i l a b i l i t y , quality, and price are very common (2). C l e a r l y there is a need for dramatic improvements in the sourcing of many important plant-based chemicals. The potential for the use of the emerging technologies of plant c e l l tissue culture for the production of valuable secondary compounds has been viewed with a sense of optimism and enthusiasm by biotechnologists. To understand the economic implications of plant tissue culture production of secondary compounds, one only has to note that, despite substantial advances in synthetic organic chemistry, plants are s t i l l the major source of twenty-five percent of a l l prescription medicines, provide the raw materials used extensively by the flavor and fragrance industries, and are the source of a number of natural sweeteners and insecticides Q). However, the i n i t i a l optimism for plant tissue culture production of secondary metabolites has been somewhat tempered by the observation that cultured plant cells routinely yield very low concentrations of the commercially most important secondary products. It is evident that the future of this area of biotechnology depends upon the development of technologies that permit the induction and selection of genetic variants that over-produce particular secondary products and the design of culture systems tailored to the unique growth requirements of plant cells. Expression of Secondary Metabolite Synthesis in C e l l Cultures Much of the d i f f i c u l t y in obtaining c e l l cultures that produce secondary products has been blamed on the lack of morphological d i f f e r e n t i a t i o n in rapidly growing c e l l cultures (t). It has been postulated that if the synthesis and subsequent accumulation of a particular secondary product is in any way dependent on specialized cellular structure, then there is no chance to exploit plant c e l l cultures for chemical production unless those s t r u c t u r a l modifications can be induced (£). Many of the most desirable secondary metabolites are formed only in highly specialized tissues, i.e. roots, leaves, flowers. For example, the cardiac glycosides of Digitalis are principally found in leaf cells; quinine and quinidine are found in the bark of Cinchona trees; and tropane alkaloids are largely synthesized in the roots and translocated to the leaves in many Solanaceae species. It has been suggested that in plant c e l l cultures this level of morphological d i f f e r e n t i a t i o n and maturation is largely absent and, therefore, secondary product synthesis is suspended. A n interesting investigation into the question of d i f f e r e n t i a t i o n versus secondary product synthesis was performed by monitoring celery flavor synthesis in celery tissue cultures (£). C e l e r y cultures at various stages of d i f f e r e n t i a t i o n , including undifferentiated cells, globular, heart, and torpedo embryos, and differentiated plants were examined for flavor compounds. The less differentiated globular and heart-shaped embryos demonstrated no flavor compounds while the more differentiated torpedo-shaped embryos, which posses chlorophyll-containing plastids, did

In Biogeneration of Aromas; Parliment, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


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possess the c h a r a c t e r i s t i c celery phlthalide flavor compounds. There were no o i l ducts in the torpedo-shaped embryos, so that these highly specialized c e l l types were not required for flavor compound production (7). It was thought that the appearance of chlorophyll and, hence, the maturation of the plastids, may in some way be associated with the formation of celery flavor compounds. A positive correlation was observed for the greening (development of chlorophyll) of celery petioles and the production of phlthalide compounds. By replacing 2,4-D in the growth medium with a structural analog, 3,5-dichlorophenoxyacetic acid, c e l l cultures of celery were isolated which began to show plastid development and chlorophyll synthesis. These green cultures also possessed phlthalide flavor compounds. Light microscopy of these c e l l cultures showed no signs of differentiation into organized meristems or embryo formation. A review on the role of morphological and cellular differentiation in the synthesis of flavor compounds has been presented by C o l l i n and Watts (8). While phlthalide biosynthesis in celery cannot be used as a general model for a l l secondary product synthesis, this example does point out the dangers in assuming that advanced morphological d i f f e r e n t i a t i o n is a prerequisite to secondary product synthesis. While greening c e l l cultures imply a more advanced plastid development than is normally found in non-green c e l l suspensions, this certainly does not represent nearly the same level of morphological differentiation previously thought necessary to initiate flavor synthesis. Therefore,itislikely that at least some c e l l cultures c h a r a c t e r i z e d as "non- producers" can be induced or "turned-on" for secondary product synthesis with relatively minor modifications in media composition. It has recently been reported that c e l l cultures of 18 of 19 species belonging to the genera Asperula, Galium, Rubia, and Sherardia produced anthraquinones at higher levels than those found in the intact plants (9). The concentration of sucrose and the types of substituted phenoxyacetic acid growth regulators were varied in an attempt to identify optimal conditions for anthraquinone production. In general, no consistent pattern of nutritional components were determined. C e l l cultures of plants from the same family, genus, or species demonstrated quite different media requirements for anthraquinone production. The dramatic effects on secondary metabolite production precipitated by alterations in the media composition provides yet another vivid example of the importance of systematically defining optimal media requirements before deeming a c e l l culture non-productive for a specific secondary compound. The use of media manipulation and potential gene expression regulators to increase overall secondary product synthesis will be discussed in a later section of this chapter. Selection of High-Producing G e n e t i c Variants Once production of the desired secondary compound is demonstrated in c e l l cultures, the emphasis can be shifted toward inducing and selecting genetic variants that synthesize increased levels of the compound. However, before one can embark on a program of genetic modification for enhanced production capacity, rapid, but sensitive assay procedures must be developed for the detection of the desired compounds (10). These methods should be geared to handle a large number of samples quickly, but should minimize the amount of tissue required for analysis.


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The selection of source material for expiants and callus initiation can be v i t a l to obtaining productive cultures. Deus and Zenk (10) stress the importance of using high-yielding differentiated plants as a source of expiant material for establishing c e l l cultures. These authors demonstrated s t a t i s t i c a l l y that high alkaloid producing plants were more likely to give rise to c e l l cultures with high alkaloid contents. Kinnersley and Dougall (11,12) have found the same relationship investigating nicotine production in c e l l cultures of Nicotiana tabacum. P l a n t Tissue C u l t u r e Biotechnology. P l a n t tissue culture biotechnology is comprised of a number of rapidly emerging and powerful technologies that can be used to: (1) reproduce identical genetic copies of e l i t e plants or plant c e l l lines, (2) generate genetic variation from expiants of cultured somatic tissue, (3) c r e a t e genetically homogeneous breeding lines v i a anther culture and the regeneration of haploids, and (4) combine desirable characteristics from two individual plants by protoplast fusion. These technologies are currently being performed with a wide range of agriculturally important crop plants and the regenerated plants are c a r e f u l l y being integrated into ongoing breeding programs. The genetic variation that is routinely observed in plants regenerated from somatic tissue has been termed somaclonal variation. Somaclonal variation can be attributed to both pre-existing genetic variation in the somatic expiants or variation induced by the c e l l culture and regeneration procedure (13). These genetic changes can be inherited by either Mendelian or non-Mendelian mechanisms and the nature of these genetic changes has been attributed to single gene mutations, chromosomal rearrangements, mitotic crossing over, and organelle mutation and segregation (1^). A review of the genetic variability in plants regenerated from somatic tissue has been presented by Reisch (14). Somaclonal variation has already proven to be an invaluable tool of the biotechnologist for introducing genetic variation into elite breeding lines. New, improved breeding lines of tomato, tobacco, oil palm, r i c e , and wheat have been obtained as the direct result of somaclonal variation programs (13,13). The most detailed c h a r a c t e r i z a t i o n of somaclonal variation has been carried out in tomato. Evans and Sharp (16) reported: (1) the recovery of 13 discrete nuclear gene mutations in different tomato breeding lines including recessive mutations for male s t e r i l i t y , jointless pedicel, tangerine-virescent fruit and flower color, chlorophyll deficiency, virescence, and mottled leaf appearance and dominant mutations for fruit ripening and growth habit; (2) that single gene mutations, derived from somaclonal variation, occur at a frequency of about one mutant in every 20-25 regenerated plants; and (3) evidence suggesting the recovery of new mutants not previously reported using conventional mutagenesis procedures. Somaclonal Variation and Secondary Product Synthesis. The induction and recovery of genetic variants by somaclonal variation technology can have a profound impact on the economic feasibility of secondary metabolite production. While most of the discussion up to this point has focused on c e l l culture production of secondary compounds, there are clearly a number of instances where whole plant production is both more e f f i c i e n t and economically prudent. This is especially true for those compounds


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that are of moderate to low cost but have large and potentially expandable markets. Compounds that f i t this scenario might be the natural sweeteners of Stevia rebaudiana, stevioside and rebaudioside, the natural pyrethrins of Chrysanthemum, special composition oils from any of the t y p i c a l specialty o i l plants like soybean or rapeseed, and essential o i l production by plants used in the flavor and fragrance industry. In these examples, successful commercialization would require very large quantities of the secondary product. While bioreactor technology for large-scale production using c e l l cultures is being developed,itisunlikely that such high levels of production in vitro would be economically feasible in the near future. Somaclonal variation can be used to induce and select new plant varieties with increased levels of accumulation for specific secondary metabolites. Interestingly, many of the genetic changes that have been attributed to somaclonal v a r i a t i o n are manifested as alterationsinthe chemical composition of the regenerated somaclone or the selfed progeny of that plant. Somaclonal variants with altered levels of carrotenoids, chlorophylls, anthocyanins, terpenes, alkaloids, and sugars are routinely observed. In p r a c t i c e , a selected somaclonal variant, with an increased level of a specific secondary metabolites, could be grown as a field crop, harvested and processed to obtain the chemical of interest. This approach represents a technically feasible and a more immediate solution t o obtaining required amounts of some low-cost, large market plant secondary compounds. However, in many cases, the more long-term approach of bioreactor production of secondary metabolites using plant c e l l cultures is more desirable for p r a c t i c a l , economic, and proprietary reasons. Plant c e l l cultures can be established from an impressive array of plant species, including most of those that produce secondary products of commercial interest (4). To date, a number of c e l l cultures have been established that produce secondary products at levels in excess of those foundinthe intact plant (Table I). However, in most instances, high-yielding lines have been described as arising spontaneously and not as the result of a tissue culture program designed to optimize the induction of genetic v a r i a t i o n . Two striking exceptions to this scenario are the selection of variants for increased nicotine synthesis in c e l l lines of Nicotiana tabacum (18) and high anthocyanin producing c e l l cultures of Euphorbia m i l l i i (19). Once the appropriate assay technique has been chosen, the production and growth media defined, and the source of expiant material determined, the process of inducing and selecting genetic variants for increased secondary product synthesis can begin (Figure 1). C a l l u s cultures, arising from somatic expiants, can be screened for chemical production and suspension cultures established from those identified as producing the desired metabolite. The advantage of using c e l l suspension cultures is that it promotes the generation of a large number of c e l l aggregates that can be replated and screened for production. The process of selecting c e l l aggregates that overproduce specific metabolites has been termed c e l l aggregate cloning (18). C e l l aggregate cloning has been used successfully to select for photoautotrophic cells (19), high vitamin-producing cells (20), high pigment cells (21), and high alkaloid containing cells (22.23) in various plant species. A n i l l u s t r a t i v e example of c e l l aggregate cloning is the selection of high anthocyanin-producing


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Table I. Secondary metabolites accumulated in high levels by plant tissue


Secondary Product Shikonin Ginsengoside Anthraquinones Ajmalicine, Serpentine Rosmarinic acid Ubiquinone-10 Diosgenin

Note:

Plant Species

PRODUCT INFORMATION C e l l C u l t u r e Plant (% of Dry Weight)

Lithospermum erythrorhizon Panax ginseng Morinda c i t r i f o l i a Catharanthus roseus

12 27 18 1.8

1.5 4.5 2.2 0.8

Coleus blumei Nicotiana tabacum Dioscorea deltoïdes

15 0.036 2

3 0.003 2

Data from references 17 and 3.

Figure 1. Somaclonal variation for development of high-producing cell lines.


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c e l l lines from cultured cells of Euphorbia m i l l i i by Yamamoto et a l (21). In these experiments, c e l l aggregates were plated on solid media and cultured. The resulting c a l l i were then divided; one half for continued growth and the other for analysis for anthocyanin content. The highest pigmented c a l l i were continually subcultured in this manner for 24 selections. The amount of pigment found in the highest producing lines after this period of time was seven times greater than the original cells. The basis for the observed chemical accumulation in some c e l l aggregates is the result of spontaneous genetic variation that was previously masked in the original expiant material or variation that was induced during the process of tissue culture initiation and subculture, i.e. somaclonal variation. Therefore, the same technology that has been successfully applied to some agricultural crop plants to select agronomically improved varieties is also widely applicable to the induction of c e l l cultures that produce high levels of secondary metabolites. The only d i f f e r e n c e between the two applicationsis,of course, the material being evaluated for genetic alteration. Somaclonal variation technology, as it is applied to crop plants, relies on the regeneration of whole plants, their s e l f - f e r t i l i z a t i o n , and the analysis of the resulting F. progeny. The application of somaclonal variation for secondary product synthesis in c e l l cultures is reliant on the generation of c e l l aggregates that can be individually selected and evaluated for specific chemical production. Genetic Stability of High-Producing C e l l Lines The genetic stability of high-producing c e l l cultures greatly a f f e c t s the economic potential of secondary metabolite production by plant tissue cultures. While stable, high-producing c e l l lines have been reported when repeated screening has been employed (21,24), this approach has been i n e f f e c t i v e in stabilizing alkaloid production in Catharanthus (17) or anthocyanin production in Daucus cultures (25). It has been suggested that instability is a function of the genetic heterogeneity of the c e l l population in a given suspension c u l t u r e (26). C e l l suspension cultures typically exist as mixtures of various c e l l types possessing a number of shapes and sizes in various degrees of aggregation. Itisexpected that this morphological variation represents c e l l types with different genetic and biochemical capacities for secondary metabolite production. A s secondary metabolite synthesis is often associated with senescent cells, the observed instability may r e f l e c t the washing out of high-producing cells due to their inherently slower growth rates relative to non-producing cells. Therefore, media compositions and c u l t u r a l p r a c t i c e s need to be tailored to enrich for producing c e l l types. Sato and Yamada (27) have recently reported the establishment of high berberine-producing c e l l cultures of Coptis japonica. It was stressed that the stability of the producing c e l l lines was highly dependent on the repeated cloning of c e l l lines that demonstrated berberine synthesis. The authors also point out the importance of c u l t u r e conditions to the maintenance of stable production levels. Fluctuations in berberine production were c o r r e l a t e d to changes in physiological conditions and the nutritional make-up of the c u l t u r e medium. It, therefore, seems likely that stable secondary metabolite synthesis is as much a function of c u l t u r a l p r a c t i c e and metabolic regulation as repeated clonal selection



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once high-producing lines have been established. The use of selective agents that favor specific c e l l types, with particular biochemical capabilities, could prove useful in maintaining stable, highly productive c e l l cultures.


Scale-Up for Secondary Metabolite

Production

Bioreactor Production of Secondary Metabolites: The scale-up of plant c e l l cultures for the production of secondary products presents a number of technical challenges. While there are some similarities between the large-scale culture of microbes and plant cells, the striking number of physiological differences between these c e l l types precludes the use of microbial fermentation systems for plant c e l l culture (28). Plant cells are generally much larger than microbial cells, and they possess rigid c e l l walls. These features render plants cells much more susceptible to the shear forces that develop in conventionl microbial blade fer mentors. Plant cells also grow much slower than microbial c e l l s , and therefore, stringent aseptic conditions need to be observed. Due to the tendency for plant cells to aggregate, settling becomes a problem in large-scale cultures in terms of oxygen transfer. Most importantly, plant cells generally do not excrete their secondary products, but retain them inside the c e l l vacuole. Therefore, a destructive harvest would be necessary to release intercellular secondary compounds. This would preclude long-term recycable c e l l cultures and increase the overall cost of production. There are a number of potential designs for plant c e l l bioreactors including: immobilized c e l l bioreactors, hollow fiber systems, a i r - l i f t vessels, and spin-filter bioreactors. Undoubtedly, no single design w i l l be sufficient for a l l applications using plant cells for secondary product synthesis. However, two systems have received a great deal of experimental attention in the area of plant secondary product synthesis: immobilized c e l l bioreactors and spin-filter bioreactors. Imobilized c e l l bioreactors exploit the physical advantages of c e l l entrappment. C e l l s have been immobilized in gels of calcium alginate (29,30), carrageenen (31), polyacrylamide, and agarose (31). The mild conditions associated with the immobilization procedure generally yields normal c e l l v i a b i l i t y (28). Indeed, plant protoplasts have been successfully immobilized in alginate-based gels (32,33). Immobilization is often reversible which allows for c e l l harvest and c e l l mass measurements. A review of plant c e l l immobilization and its uses for plant secondary product synthesis is presented by Brodelius (34). Immobilized plant cells have respiration and bioconversion rates that are very similar to plant cells in suspension cultures but also have the advantage of the physical protection of the immobilizing matrix (28). The e f f e c t s of the shearing forces created by the movements of the media is greatly reduced in immobilized systems. C e l l immobization also permits the operation of a continuous culture bioreactor at dilution rates in excess of the maximum growth rate of the culture as entrapped cells are not as susceptible to washout as those in batch cultures. Immobilized cells are often set up in a fluidized-bed configuration or a packed-bed system. Both systems have been described in a review by Prenosil and Pedersen (28). The ease in which the media can be c i r c u l a t e d through the immobilized c e l l bed and c o l l e c t e d has led several investigators to this system for biotransformation studies. Indeed, the conversion of


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inexpensive precursors to the more valuable end-product by immobilized cells has been studied for a number of secondary products listed in Table II. A detraction of the immobilized c e l l reactors is the requirement that the plant c e l l must excrete its secondary compounds. T h e o r e t i c a l l y , the production media would flow across the c e l l bed providing nutrition to the cells and removing the chemical products. A t some point, the medium would be exchanged and the desired product extracted from the spent medium. However, plant cells do not normally excrete secondary products into the medium, but rather, they sequestor these compounds in the vacuole. Studies on plant c e l l excretion have suggested that accumulation and excretion of secondary products may be conveniently modulated by s t r i c t c o n t r o l of external pH (40). P r e c i s e definition of specific excretion and accumulation parameters for individual c e l l lines would undoubtedly prove invaluable in designing c o s t - e f f e c t i v e bioreactors for scale-up of producing c e l l cultures. Recently, non-destructive permeabilization of immobilized plant cells by treatment with organic chemicals has been reported (41). Another potentially e x c i t i n g system for plant secondary product synthesis is the spin-filter bioreactor. O r i g i n a l l y designed for mammalian c e l l culture (42), the spin-filter bioreactor permits continuous c u l t u r e of plant cells at very high densities without c e l l washout. This is accomplished by the use of a spinning f i l t e r which allows for the removal of spent media and the introduction of fresh media without washing out c e l l mass. The f i l t e r rotates so as to prevent clogging, but the rate of rotation is slow enough to avoid c e l l damage. Therefore, this design f a c i l i t a t e s increased nutrient feed rates without reducing the c e l l growth rate, a significant difference over conventional batch cultures. One drawback of the spin-filter bioreactor might be the formation of c e l l aggregates. The inner most cells of the c e l l aggregate are distinctly d i f f e r e n t from those on the outer edge (28,43) Metabolic release of chemicals also varies between single cells and aggregates as well as within the aggregate itself. It has been shown that single cells are a more reliable source of chemicals, therefore, the accumulation of c e l l aggregates must be controlled. It has been observed that the ratio of c e l l types within a culture can be controlled by the degree of physical mixing within the bioreactor. The use of air spargers and modified paddle-type impellers limit the accumulation of aggregates by providing aeration and a g i t a t i o n . Whatever the specific design of the bioreactor, it is likely that tissue culture biotechnology can be employed to further increase secondary product synthesis in high-yielding c e l l lines. These manipulations can have significant impact on the economic feasibility of plant tissue culture production of secondary metabolites, and in many cases, are specifically directed to the technical problems associated with the scale-up of plant c e l l cultures. Production Medium. It is v i t a l l y important to define a culture medium that promotes the production of the secondary metabolite of interest. As a production medium is unlikely to support the level of growth required to obtain the appropriate biomass, it may be desirable to develop a two-step approach whereby one medium is utilized solely for growth, and a second is employed for secondary product synthesis and accumulation (44). The



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Table II. Biotransformation for the production of plant secondary metabolites.

Plant Species Digitalis lanata Daucus c a r o t a Solanum tuberosum Papaver somniferum Citrus

Substrate digitoxin digitoxigenin solavetivone thebaine codeinone valencene

Product

Reference

digoxin hydroxylated derivatives sequiterpene neopine codeine nootkatone


29,30

35 36 37 38 39

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e f f e c t of altering the basic components of plant tissue c u l t u r e media on the production of secondary compounds has been extensively reported in the literature (45,46). When formulating a production medium, one must consider the dramatic e f f e c t s on secondary product synthesis incurred by altering even minor components of the medium. Carbon source (sucrose, glucose, fructose, etc.), nitrogen source (organic or inorganic), vitamins, and ions have a l l been shown to play significant roles in altering the expression of secondary metabolic pathways. The presence or absence of phosphate ions plays an important role in the expression and accumulation of some secondary products. Zenk et al. (47) have demonstrated a 5 0 % increase in anthraquinone accumulation in c e l l cultures of Morinda c i t r o f o l i a when phosphate was increased to a concentration of 5g7E ïrTsuspension cultures of Catharanthus roseus, the overall accumulation of secondary metabolites like tryptamine and indole alkaloids has been shown to occur rapidly when cells were shifted to a medium devoid of phosphate (48,49). A study on the uptake of phosphate and its e f f e c t on phenylalanine ammonia lyase and the subsequent accumulation of cinnamoyl putrescine in c e l l suspension cultures of Nicotiana tabacum demonstrated marked sensitivity to phosphate concentration (iQ). Enhanced phenylalanine ammonia lyase a c t i v i t y and increased production of cinnamoyl putrescine was induced by subculture onto phosphate-free medium while suppression of these e f f e c t s and stimulation of growth was observed with phosphate concentrations of 0.02-0.5uM. Interestingly, phenylalanine ammonia lyase a c t i v i t y is stimulated by increasing phosphate concentrations in c e l l suspension of Catharanthus roseus 01)· An additional insight into the importance of specific media components on production of secondary products can be gained by examining the case history of shikonin production. It had been shown that callus cultures of Lithospermum erythrorhizon could be induced to produce shikonin on Linsmaier-Skoog medium supplemented with ΙμΜ indole a c e t i c acid (IAA) and ΙΟμΜ kinetin (KIN) (52). The e f f e c t s of specific nutritional components of the tissue culture medium on growth of the c e l l cultures and production of shikonin were also investigated X53). Fujita et a l . (54,55) found that the levels of N0 ", C u , and SO^~ had profound e f f e c t s of shikonin biosynthesis. Optimal concentrations were identified for each ion (18) as well as optimal levels of key organic components. The resultant medium supported production of shikonin at a rate approximately 13 times that obtained on previous media formulations. + +

3

Analogs. Synthesis of chemical compounds has been induced in c e l l suspensions by the addition of structural analogs. For instance by substituting an analog for the natural amino acid in a biosynthetic pathway, metabolic a c t i v i t y in that particular biosynthetic pathway can be increased or decreased. The amino acid analog 4-methyltryptophan, has been used to select Cartharanthus roseus c e l l lines that produce increased concentrations of tryptamine (56). This is accomplished by relieving feedback inhibition c o n t r o l over the tryptophan biosynthetic pathway thus increasing carbon flow toward tryptamine synthesis. These cells also overproduce the alkaloid, ajmalicine. Though successful chemical production increases have been attained using a number of amino acid analogs, many high producing c e l l lines have proven genetically unstable,


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and continuous selection is required t o maintain increased production rates (56). Growth Regulators. Phytohormones and other growth regulating compounds have long been known to influence morphological and, therefore, physiological development of cultured plant cells or whole plants. The commonly used phytohormone analog, 2,4-D has been implicated in a number of systems as interfering with secondary metabolism (57). In carrot c e l l cultures the presence of exogenous giberellic a c i d blocks anthocyanin synthesis by preventing chalcone-biosynthesis (58). The chemical agent 2-diethylaminoethyl2,4-dichlorophenylether and its derivitives have proven t o have a profound e f f e c t on alkaloid production in C. roseus. A 2 0 % increase in total alkaloids with a similar increase in ajmalicine, and catharanthine was noted (1,52). Understanding hormonal influences at the molecular level w i l l be necessary t o unlock the secret of these powerful regulators and to use them to s p e c i f i c a l l y modulate gene a c t i v i t y . M i c r o b i a l Insult/Fungal E l i c i t o r s . Itiscommon in nature to find that microbial insult of whole plants leads to the production of specific secondary metabolites. The molecules responsible for stimulating secondary product synthesis are referred to as elicitors. Fungal elicitors are the best studied e l i c i t o r s , and their a c t i v e regulatory molecules have been identified as being glucan polymers, glycoproteins, and low molecular weight organic acids (59). Albershiem and his colleagues refer to these regulatory molecules as oligosaccharins (60-.61). A n example of an elicitor inducing a latent biosynthetic capability is found in parsley, where synthesis of the coumarin compound psoralen has been induced by fungal elicitors (62). Derivatives of psoralen are used in the treatment of psoriasis and as ingredients of photosensitizing suntan lotions. Treatment of parsley cells by the addition of a c e l l w a l l f r a c t i o n of the fungus Phytopthora megasperma f. sp. glycinea resultedinthe production of the mRNA's encoding two enzymes of phenylpropanoid metabolism, namely phenylalanine ammonia-lyase (PAL) and 4-coumarate (CoA-ligase) (£2). Another instance of the u t i l i t y of fungal elicitors is the use of autoclaved fungal mycelia to increase yields of diosgenin in c e l l suspensions of Mexican Yam (64). Diosgenin is a steriodal saponin, an important precursor in the preparation of oral contraceptives and other medical steroids. C l e a r l y , fungal elicitors have become an a t t r a c t i v e tool for regulating secondary product synthesis. Fungi can be easily grown, recovered, and harvested, and crude preparations can be conveniently screened for inducing chemical synthesis in cultured cells (3fL). P h y s i c a l Stress. P h y s i c a l stress factors such as temperature variations, pH change, and light exposure have in some cases resulted in inhibition of chemical production and in other systems stimulated secondary synthesis. C e l l suspensions of Papavar bracteatum grown at 36 C have been shown to accumulate protopine, sanguinarine, isothebaine, and orientalidine. Reducing this temperature to 17 C caused growth inhibition and a further reduction to 5 , caused the release of thebaine into the media. A s chilling stress is known to severely inhibit photosynthetic reactions and Q


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disrupt organelles (41), the thebaine was most likely produced during growth inhibition and released due to the stress caused by chilling (66).


Light induced synthesis of secondary metabolites has been exploited in several c e l l cultures. The previously described e f f e c t s of fungal elicitors that induce mRNA production encoding for P A L and CoA-ligase in parsley, is duplicated when these cells are irradiated with UV light (63). Anthocyanin synthesis in many plant tissues has long been known to be promoted by light. More recent work has revealed that red light and far-red light accentuates and reduces, respectively, anthocyanin synthesis in apple fruit skin and poinsetta. Itisevident that a phytochrome is involved in a reversible reaction in regulating anthocyanin (67) synthesis. Protoplast Fusion for Fine-Tuning Producing Cultures: Although much more speculative than the application of somaclonal variation for the induction and selection of high-yielding c e l l lines or whole plants, the technology of protoplast fusion may play a v i t a l role in genetically fine-tuning c e l l cultures for increased production. Where the addition of exogenous phytohormones have been shown to impair secondary metabolism, fusion of protoplasts from the producing c e l l line with protoplasts of a hormone autotrophic tumor c e l l might result in fusion products that demonstrate growth and increased production in a hormone-free medium. A n intuitively appealing idea is the prospect of culturing photoautotrophic cells for secondary metabolite production. These cultures would have the economic advantage of using solar energy d i r e c t l y (i.e. photosynthesis) and would, therefore, be capable of growth and production on a medium with l i t t l e or no exogenous sugar. The scale-up of c e l l cultures for the production of secondary compounds w i l l require aseptic conditions for prolonged periods of time. A s plant tissue culture media is relatively expensive and inviting to microbial contamination, any modification that would reduce both cost and contamination problems would have significant impact on the feasibility of tissue culture production of secondary metabolites. Selection procedures for isolation of photoautotrophic c e l l lines have been reviewed by Yamada and Sato (68). It should be noted that the culture of photoautotrophic cells has the potential for increasing secondary product synthesis. In instances where the synthesis of a particular secondary metabolite is regulated by the level of cellular d i f f e r e n t i a t i o n , it is anticipated that the culture of photoautotrophic cells w i l l have significant e f f e c t s on production. Indeed, some alkaloids (69) vitamins (20), and components of volatile essential oils (70) have been produced by cultured green cells. Photoautotrophy may be introduced into a producing c e l l line by either of two methods: (a) producing c e l l lines can be selected for photoautotrophy, or (b) protoplasts from a photoautotrophic c e l l line can be fused with protoplasts of the producing c e l l line and selection for both c h a r a c t e r i s t i c s performed on subsequent fusion products. Concluding

Remarks

The rapidly emerging technologies of plant tissue culture biotechnology are poised to have a significant impact on the production of valuable


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secondary metabolites by plant cell culture or whole plants. Somaclonal variation has proven to be a powerful tool for inducing useful genetic variants for the improvement of agriculturally important crop plants. In a similar fashion, somaclonal variation technology can be used to induce high-yielding cell cultures. Once high producing variants have been selected, precise definition of growth and production media will further increase production and facilitate the maintenance of stable producing cultures. These advancements will eventually permit the cost-effective, industrial scale production of plant secondary metabolites.


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April 4, 1986


Production of Secondary Metabolites in Plant Cell Cultures - American

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